The potential threat of an intentional release of chemical nerve agents along with thousands of fatalities in developing countries every year caused by pesticide poisoning has made treatments for these types of poisoning a persistent focus (Eddleston, QJM: Monthly Journal of the Association of Physicians, 93:715-731 (2000); Phillips, et al., Lancet, 359: 835-840 (2002)). Many pesticides and chemical nerve agents rely on the same organophosphate core to inhibit acetylcholinesterases. For example, G-type nerve agents such as soman (o-pinacolyl methylphosphonofluoridate) possess the same fluorophosphate moiety as the pesticide mipafox (N,N′-Diisopropyldiamidofluorophosphate; DDFP). In the case of V-type nerve agents, the fluoride on the organophosphate core is substituted with a thiol group (Coulter, et al., U.S. Army Chemical Warfare Laboratories Technical Report CWLR, 2346 (1959)). Current treatments for organophosphate poisoning involve a combination of atropine (a muscarinic antagonist), pralidoxime (also known as 2-PAM, a reactivator of poisoned acetylcholinesterase enzymes) and benzodiazepines to control seizures (Buckley, et al., BMJ, 329:1231-1233 (2004)).
Another method of treating organophosphate poisoning has recently emerged through the potential use of catalytic enzymes to detoxify these poisons in the blood. Detoxification of these poisons offers the particular advantage of altering organophosphates before they enter the tissue where they bind acetylcholinesterase in the neuromuscular junctions. Therefore, such enzymes can at a minimum work in a complementary manner to, and in concert with, other treatments to improve survival rates. This co-treatment was demonstrated when a liposome-encapsulated diisopropylfluorophosphate (DFP)—hydrolyzing enzyme, known as an organophosphorus acid anhydrolase (OPAA) was used in conjunction with atropine and 2-PAM to confer 25 LD50s of protection against diisopropylfluorophosphate (Petrikovics, et al., Toxicological Sciences: An Official Journal of the Society of Toxicology, 57:16-21 (2000)).
OPAA (EC 3.1.82) is one example of a larger class of enzymes known as phosphotriesterases, initially identified over sixty years ago and generally recognized for their ability to detoxify organophosphates (Bigley, et al., Biochimica et Biophysica Acta, 1834:443-453 (2013)). Phosphotriesterase (PTE), methyl parathion hydrolase (MPH), diisopropylfluorophosphatase (DFP) and paraoxonase 1 (PON1) are the other examples from this class (Bigley, et al., Biochimica et Biophysica Acta, 1834:443-453 (2013)). These enzymes are all hydrolases with hydrophobic active sites and a requirement for divalent metals but they differ in terms of sequence, structure and their catalytic mechanisms. The native activity of OPAA is that of a prolidase (Cheng, et al., Journal of Industrial Microbiology & Biotechnology, 18:49-55 (1997)). The OPAA enzyme from the gram-negative, aerobic, short rod bacterium Alteromonas sp. JD6.5 has drawn particular attention because it has very high activity on GD as well as a very high level of expression in E. coli (DeFrank, et al., Chemico-Biological Interactions, 87:141-148 (1993); DeFrank, et al., Journal of Bacteriology, 173:1938-1943 (1991)). However, it has not been reported to have any activity against V-type nerve agents.
An effective broad-spectrum enzymatic antidote addressing nerve agent poisoning would need to include enzymes with good catalytic efficiency (target value of about 107 min−1 M−1) on as many relevant substrates as possible. Regrettably, the activity of the wild-type OPAA enzyme on VR (approximately 5×102M−1 min−1) falls well short of what is necessary for use in that capacity.
Furthermore, with enzyme-substrate interactions more often than not being very stereospecific, enhancing the catalytic activity of OPAA may only be one component necessary to achieve an effective catalytic antidote. Previous reports have already indicated that the toxicity of chemical nerve agents can be extremely stereospecific, as evidenced by the case of soman (o-pinacolyl methylphosphonofluoridate), where the most toxic C(−)P(−) enantiomer is more than 100 times as toxic as the least toxic C(+)P(+) enantiomer, corresponding to mouse LD50 values of 38 μg/kg vs >5000 μg/kg, respectively (Benschop, et al., Toxicology and Applied Pharmacology, 72:61-74 (1984)). The differences in toxicity generally correspond to the different affinities with which the respective enantiomers bind acetylcholinesterase (Benschop, et al., Toxicology and Applied Pharmacology, 72:61-74 (1984)), although they may also be attributable to differential detoxification in vivo. For example, native enzymes such as human paraoxonase may preferentially catalyze the less toxic P(+) enantiomers of soman (Yeung, et al., Journal of Analytical Toxicology, 32:86-91 (2008)), thereby reducing the measure of their toxicity. Generally, it is known that the action of catalytic enzymes on nerve agents occurs in a stereospecific manner, both with G-agents (Harvey, et al., Enzyme and Microbial Technology, 37:547-555 (2005); Tsai, et al., Biochemistry, 51:6463-6475 (2012)) and with VX (Bigley, et al., Journal of the American Chemical Society, 135:10426-10432 (2013)). Although OPAA is easily produced at high levels and is stable in normal use, its activity against V-type chemical agents is extremely low and it shows an almost absolute preference for the less toxic enantiomers of soman.
Understandably, an effective enzymatic, broad-spectrum antidote to nerve agent poisoning would need to include enzymes that possess not only good catalytic efficiency on racemic materials, but activity specifically directed towards the toxic isomers of all relevant substrates. This combination of catalytic efficiency coupled with the proper stereochemistry was achieved recently with the H257Y/L303T mutant of the bacterial phosphotriesterase (PTE) enzyme for the substrates sarin, soman and cyclosarin (Tsai, et al., Biochemistry, 51:6463-6475 (2012)). The mutant enzyme possessed catalytic efficiencies approximately ten times greater than wild-type PTE on sarin and cyclosarin and approximately 100 times greater on soman, as well as a reversal of stereospecificity so that the mutant possessed greater activity on the more toxic P(−) isomer than the P(+)isomer (the wild-type enzyme prefers the P(+) isomer).
VX (O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate), which is a structural isomer of VR, has about a 13-fold difference in toxicity between its enantiomers, with the P(−) isomer being the more toxic of the two (Hall, et al., The Journal of Pharmacy and Pharmacology, 29:574-576 (1977)). Given that VX and VR are structural isomers, have similar racemic toxicities (Benschop, et al., Toxicology and Applied Pharmacology, 72:61-74 (1984); Hall, et al., The Journal of Pharmacy and Pharmacology, 29:574-576 (1977)), and trends in cholinesterase inhibition constants with the respective enantiomers (the P(−)enantiomers of each are much more inhibitory); (Bigley, et al., Journal of the American Chemical Society, 135:10426-10432 (2013)), and that both are treated with similar modalities using acetylcholinesterase reactivators (Kuca, et al., Basic & Clinical Pharmacology & Toxicology, 98:389-394 (2006)), it is likely that the relative toxicity of their respective enantiomers follows a similar pattern. Therefore, there remains a need for improved OPAA enzymes.
It is an object of the invention to provide engineered OPAAs with increased catalytic efficiency for organophosphates, particular V-type nerve agents such as VX and VR.
It is a further object of the invention to provide engineered OPAAs with broadened stereospecificity capable of catalysis of both enantiomers of V-type nerve agents such as VX and VR.
It is also an object of the invention to provide methods of using engineered OPAAs to treat and inhibit nerve agent poisoning.
It is another object of the invention to provide methods of using engineered OPAAs in various detoxification applications.